Hepatitis C is one of the most widespread infectious diseases in the world. About 180 million people are infected with hepatitis C virus (HCV) worldwide with a yearly incidence of 3-4 million. While the acute phase of infection is mostly asymptomatic, the majority of acutely infected individuals develops chronic hepatitis and is at increased risk of developing liver cirrhosis and hepatocellular carcinoma. Thus, HCV infection is a major contributor to end-stage liver disease and in developed countries to liver transplantation.
HCV is a small, enveloped virus classified as a member of the Flaviviridae family. Its genome consists of a 9.6 kb single stranded RNA of positive polarity composed of 5′ and 3′ untranslated regions (UTR) and one long open reading frame (ORF) encoding a polyprotein, which is co- and post-translationally cleaved and thus yields the structural (Core, E1, E2), p7 and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, NS5B) proteins.
HCV isolates from around the world exhibit significant genetic heterogeneity. At least 7 major HCV genotypes (genotypes 1-7) have been identified, which differ by 31-33% at the nucleotide level and deduced amino acid level. In addition, there are numerous subtypes (a, b, c, etc.), which differ by 20-25% on the nucleotide and deduced amino acid level.
While HCV genotypes 1-3 predominate in the Western World, genotypes 4-6 are more common in areas with high prevalence or even endemic levels of HCV infection. Genotype 6 is highly prevalent in Southeast Asia. Recently, a genotype 7a was discovered in Canadian and Belgian patients, who presumably were infected in Central Africa (Murphy et al., 2007). Thus, the knowledge about the natural history, susceptibility to treatment and neutralizing antibodies as well as receptor interactions of this new genotype is very limited.
While the only approved treatment for chronic HCV infection, combination therapy with interferon-α and ribavirin, leads to a sustained virologic response in most of genotype 2 or 3 patients, viral clearance is only obtained for about half of patients with genotype 1 or 4. There is nothing known about treatment responses of HCV genotype 7a infected patients. There is no vaccine against HCV.
Since its discovery in 1989, research on HCV has been hampered by the lack of appropriate cell culture systems allowing for research on the complete viral life cycle as well as new therapeutics and vaccines.
In 2001, a genotype 2a isolate (JFH1) was described (Kato et al., 2001), which yielded high RNA titers in the replicon system without adaptive mutations (Kato et al., 2003).
A major breakthrough occurred in 2005, when formation of infectious viral particles was reported after transfection of RNA transcripts from the JFH1 full-length consensus cDNA clone into Huh7 cells (Wakita et al., 2005) (Zhong et al., 2005).
At the same time, Lindenbach et al. demonstrated that the intragenotypic 2a/2a recombinant genome (J6/JFH1), in which the structural genes (Core, E1, E2), p7 and NS2 of JFH1 were replaced by the respective genes of clone J6CF, produced infectious viral particles in Huh7.5 cells (a cell line derived from bulk Huh7 cells) with an accelerated kinetic (Lindenbach et al., 2005). Cell culture derived J6/JFH viruses were apparently fully viable in vivo.
Despite the importance of the described cell culture systems they represent only a single subtype (genotype 2a) of HCV. It is important to develop cell culture systems for representative strains of other HCV genotypes, since neutralizing antibodies are not expected to cross-neutralize all genotypes and new specific antiviral compounds might have differential efficiencies against different genotypes. For the genotype specific study of the function of the structural proteins, p7 and NS2 as well as related therapeutics such as neutralizing antibodies, fusion inhibitors, ion-channel blockers and protease inhibitors, it would be sufficient to construct intergenotypic recombinant viruses in analogy to J6/JFH.
Pietschmann et al. 2006 disclose construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus recombinants. The authors created a series of recombinant genomes allowing production of infectious genotype 1a, 1b, 2a and 3a particles by constructing hybrid genomes between the JFH1 isolate and the HCV isolates: H77 (genotype 1a), Con1 (genotype 1b), J6 (genotype 2a) and 452 (genotype 3a) respectively. Thus, disclosing genotypes completely different from the genotype disclosed in the present application.
The infectious titers of the 1a, 1b and 3a genotypes disclosed in Pietschmann et al. 2006 are not at a level sufficiently high for practical utilization in functional analysis, drug and vaccine development or other applications. For such applications, including screening of potential drugs and development of potential vaccine candidates, the skilled person will know that infectivity titers below 103 TCID50/mL contain insufficient amounts of infectious virus.
Accordingly, the study does not attempt cell culture adaptation of the genotype recombinants, e.g. by serial passage of cell culture derived viruses to naïve cells and it is not investigated whether adaptive mutations develop after transfection in cell culture. In fact, Pietschmann et al does not even provide any sequence data of the virus produced in the cell culture.
In D1 (WO 2006/096459), Rice et al. discloses intragentoypic and intergenotypic chimeric HCV genomes comprising Core, E1, E2, p7 and NS2 from a first HCV strain and 5′UTR, NS3, NS4A, NS4B, NS5A, NS5B and 3′UTR of a second HCV strain. As experimental work backing this claim is presented the following: 1) Generation and characterization of the intragenotypic recombinant J6/JFH1 (with Core-NS2 of genotype 2a strain J6 as well as 5′UTR, NS3-NS5B and 3′UTR of genotype 2a strain JFH1), 2) Generation and characterization of the intergenotypic recombinant H77/JFH1 (with Core-NS2 of genotype 1a strain H77 as well as 5′UTR, NS3-NS5B and 3′UTR of genotype 2a strain JFH1). Further, adaptive mutations are identified in H77/JFH1.
In D2, Gottwein et al. describe the generation and characterization of the intergenotypic HCV genome S52/JFH1 (with Core-NS2 of genotype 3a strain S52 as well as 5′UTR, NS3-NS5B and 3′UTR of genotype 2a strain JFH1). Adaptive mutations that are necessary for efficient growth in cell culture are identified and tested in reverse genetic studies. Optimally cell culture adapted S52/JFH1 genomes are constructed and characterized. Applicability of this genotype 3a/2a cell culture system is shown in receptor blocking studies (blocking of the putative HCV receptor CD81) and confocal microscopy studies, investigating co-localization of HCV Core with intracellular lipids.
In D3, Scheel et al. describe the generation and characterization of the intergenotypic HCV genome ED43/JFH1 (with Core-NS2 of genotype 4a strain ED43 as well as 5′UTR, NS3-NS5B and 3′UTR of genotype 2a strain JFH1). Adaptive mutations that are essential for viability in cell culture are identified and tested in reverse genetic studies. Optimally cell culture adapted ED43/JFH1 genomes are constructed and characterized. Applicability of this genotype 4a/2a cell culture system is shown in receptor blocking studies (blocking of the putative HCV receptor CD81) and neutralization studies.